Coxiella burnetii is a ubiquitous zoonotic bacterial pathogen and the cause of human acute Q fever, a disabling influenza-like illness. Coxiella's former obligate intracellular nature significantly impeded genetic characterization of putative virulence factors. However, our seminal advance of host cell-free (axenic) growth of Coxiella in acidified citrate cysteine medium (ACCM) enabled us to quickly develop shuttle vector, transposon mutagenesis, inducible gene expression, targeted gene deletion, and nutritional selection technologies for pathogen genetic manipulation. We also developed a Luciferase gene reporter system that can be used to monitor developmentally- regulated gene expression and pathogen stress responses. The repertoire of Coxiella genetic tools now allows traditional mutation and complementation strategies for virulence factor discovery. Indeed, we have constructed knockout strains in both virulent and avirulent Coxiella, including those with deletions in genes encoding components of the Dot/Icm type IVB secretion system (T4BSS) and secreted proteins. These studies have confirmed that T4BSS function is critical for Coxiella growth in macrophages. Mutational analysis has also identified several T4BSS effector proteins that are required for optimal growth in mammalian cells. Coxiella encodes a paucity of transcriptional regulators that are likely critical for intramacrophage survival and/or developmental transitions. The GacAS two-component systems (TCS) of Coxiella are especially intriguing as homologous systems in other bacteria regulate complex social behaviors, virulence gene expression, and developmental transitions. Moreover, the role the stationary phase sigma factor RpoS in stress survival is unknown. Unraveling GacAS and RpoS regulatory networks will identify important virulence determinants. Using gene knockouts, reporter assays, RNAseg, and whole bacterial proteome mass spectrometry, we aim to resolve regulatory cascades governing infectivity, developmental form changes, and stress responses. These studies will provide needed insight into Coxiella virulence. The only genetic lesions proven to result in attenuated Coxiella virulence in an immunocompetent animal model are associated with defective lipopolysaccharide (LPS) synthesis. Virulent phase I organisms with full-length LPS transition to avirulent phase II organisms with severely truncated LPS upon repeated in vitro passage. Given the critical role of LPS in Coxiella virulence, it is important to understand the molecular basis of phase variation. Whole genome sequencing of high passage phase II strains in our collection has revealed indels (insertions/deletions) associated with phase variation. Based on these data, allelic exchange and complementation experiments are being conducted to genetically define pathways of phase conversion. Coxiella undergoes an intracellular biphasic developmental cycle that generates two distinct morphological variants that can be distinguished by ultrastructure and protein composition. Small cell variants (SCV) do not replicate, contain condensed chromatin, and are considered extracellular survival forms. SCV differentiate into replicative large cell variants (LCV) with dispersed chromatin. Transition of LCV back to SCV occurs coincident with entry of Coxiella into stationary growth phase with nearly homogeneous SCV present upon extended incubation (2 to 4 weeks) of infected cell cultures. As an amenable model to help us better understand the biological relevance of Coxiella differentiation, we established that SCV/LCV transitions are recapitulated by organisms growing in the third-generation axenic media, ACCM-D. This discovery enables studies of Coxiella developmental biology without experimental difficulties encountered with host cell-propagated bacteria. Comparative transcriptomics and proteomics of LCV and SCV have now revealed molecular determinants of morphological differentiation that likely contribute to the unique biological characteristics of cell forms. Genes associated with differentiation are now being inactivated and mutants phenotyped.